Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics.

We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli.

T-cell determinants and antibody binding sites on the major mycobacterial secretory protein MPB59 of Mycobacterium bovis.

Among the first proteins encountered by the host immune system upon infection or vaccination with mycobacteria are those secreted by the bacillus during growth. The antigen 85 complex of Mycobacterium bovis bacillus Calmette-Gúerin (BCG) is composed of three closely related members. The mature 85B protein of M. bovis (MPB59) has a high degree of amino acid identity with the M. bovis 85A protein (76%) and the Mycobacterium tuberculosis 85B (99%) and 85A (76%) proteins. We have examined the regions of MPB59 which stimulate human T- and B-cell responses by use of a set of 28 synthetic peptides, 20 amino acids (aa) in length and overlapping by 10 aa. Initial proliferative assays with recombinant MPB59 demonstrated that peripheral blood mononuclear cells from 95% of BCG vaccinees and 52% of tuberculosis patients responded to the whole mature protein. Peripheral blood mononuclear cells from MPB59 responders, but not nonresponders, were stimulated by peptides in a dose-dependent fashion. Five peptides were reactive in more than half of the MPB59 responders. The T-cell-reactive regions were essentially identical in the M. bovis and M. tuberculosis 85B proteins. Subjects with a variety of HLA-DR phenotypes responded to a number of these peptides. The dominant T-cell-reactive regions were distinct from the peptides recognized by sera from tuberculosis patients (aa 71 to 100) and the murine monoclonal antibody HYT27 (aa 61 to 90). The region reactive with antibodies overlapped part of the MPB59 sequence recently shown to participate in the binding of MPB59 to fibronectin.

Identification of a Mycobacterium leprae-specific T cell epitope on the 70 kDa heat shock protein.

A major antigen of the leprosy bacillus, Mycobacterium leprae, is the 70 kDa heat shock protein (Hsp70), which has significant sequence homology with Hsp70 from other mycobacterial species as well as Hsp70 from eukaryotes. A unique region of 70 amino acids at the C-terminus of the M. leprae Hsp70 has been previously identified. This study investigated whether mice immunized with the C-terminal fragment of M. leprae Hsp70 recognize T cell epitopes in this species-specific portion of the molecule. Murine lymphoproliferative responses to overlapping peptides spanning the C-terminal 70 amino acids were restricted to mice of an H-2b haplotype and identified the presence of a determinant in sequence 567-591. Lymph node cells from mice immunized with this peptide recognized both the C-terminal fragment and the whole Hsp70 molecule. Moreover, mice immunized with the same peptide responded to the whole Hsp70 molecule in a delayed-type hypersensitivity reaction. The significance of M. leprae-specific T cell epitopes in the host response to mycobacterial infection is discussed.

Mechanism of interaction of the 85B secreted protein of Mycobacterium bovis with fibronectin.

The 85B protein of Mycobacterium bovis is a member of the secreted antigen 85 complex, which has been identified in a number of pathogenic mycobacteria. The 85 complex contains three components with molecular masses of 30 to 32 kDa which share the property of binding to fibronectin, a large glycoprotein present in plasma. To investigate this activity we have expressed the M. bovis 85B antigen as a recombinant protein and studied its interaction with human fibronectin. Fibronectin bound to the immobilized 85B protein in a solid-phase enzyme-linked immunosorbent assay (ELISA) and in the fluid phase in a radioimmunoassay using 125I-labelled 85B protein. In addition, fibronectin reacted with immobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subsequently identified by N-terminal sequencing. These fragments contained fibronectin-reactive peptides identified in ELISAs utilizing a set of 28 overlapping 20-mer peptides encompassing the 85B sequence. Further studies showed that the 85B protein reacted with a 32-kDa polypeptide from a limited tryptic digest of fibronectin which was identified as the collagen-binding domain. This region was confirmed as the 85B binding site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin interaction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.

Analysis of B-cell epitopes in the variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein.

The C-terminal region of the Mycobacterium leprae 70-kDa heat-shock protein is the major target for the humoral immune response to this protein and contains M. leprae-specific sequences. To examine B-cell responses to this region more closely, we constructed and expressed a recombinant fragment of the M. leprae P70 gene that encodes the C-terminal 142 residues (C-142) and synthesized a series of 10 overlapping peptides to encompass this region. The affinities of three monoclonal antibodies (MAbs) reactive with this region of P70 were measured, and the binding site of the highest-affinity MAb was determined to lie between residues 498 and 515. This reactivity was confirmed by a fluid-phase inhibition enzyme-linked immunosorbent assay. By contrast, sera from leprosy patients which were strongly reactive with the C-142 fragment failed to bind directly to the conjugated or unconjugated peptides. To determine whether the M. leprae-specific C-terminal 70 residues could stimulate B-cell responses, the reactivity of hyperimmune anti-M. leprae P70 antisera with the peptides was examined. Rabbit polyclonal anti-M. leprae P70 antisera recognized epitopes between residues 498 and 515 and in the M. leprae-specific region between residues 567 and 591. The latter, in turn, when coupled to ovalbumin, was able to generate a strong anti-P70 response specific for mycobacterial, but not human, HSP70. Three strains of mice immunized with either C-142 or P70 recognized epitopes in the region between residues 487 and 532, but the response varied with the strain and immunogen. These data demonstrate that two regions in the C-terminal portion of M. leprae P70 contain linear B-cell epitopes recognized by MAbs or hyperimmune serum. Sera from leprosy patients, however, react predominantly with conformational determinants in the immunodominant C-terminal part of the protein.

Characterization of the functional properties of the 70-kDa protein of Mycobacterium bovis.

A number of mycobacterial proteins have been shown to induce strong humoral and cellular immune responses, including the 70-kDa antigen (p70) of Mycobacterium leprae and Mycobacterium bovis. On the basis of sequence homology and an ATP binding ability, p70 has previously been tentatively allocated to the 70-kDa family of heat shock proteins (hsp70). We have purified the M. bovis p70 antigen and described ATPase and Ca(2+)-dependent autophosphorylating activities. These co-purified with p70 on gel chromatography and were up-regulated by native proteins and down-regulated by peptides. Inhibitory peptides were shown to bind p70. These data imply close functional similarities of mycobacterial p70 to other members of the hsp70 family, the Escherichia coli homologue dnaK in particular.